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Aim: This study aimed to assess intracellular Ca 2+ dynamics in nerve cells and Schwann cells in isolated rat resistance arteries and determine how these dynamics modify noradrenaline release from the nerves and consequent force development., Methods: Ca 2+ in nerves was assessed with confocal imaging, noradrenaline release with amperometry and artery tone with wire myography. Ca 2+ in axons was assessed after loading with Oregon Green 488 BAPTA-1 dextran. In other experiments, arteries were incubated with Calcium Green-1-AM which loads both axons and Schwann cells., Results: Schwann cells but not axons responded with a Ca 2+ increase to ATP. Electrical field stimulation of nerves caused a frequency-dependent increase in varicose [Ca 2+ ] ([Ca 2+ ] v ). ω-conotoxin-GVIA (100 nmol/L) reduced the [Ca 2+ ] v transient to 2 and 16 Hz by 60% and 27%, respectively; in contrast ω-conotoxin GVIA inhibited more than 80% of the noradrenaline release and force development at 2 and 16 Hz. The K V channel blocker, 4-aminopyridine (10 µmol/L), increased [Ca 2+ ] v , noradrenaline release and force development both in the absence and presence of ω-conotoxin-GVIA. Yohimbine (1 µmol/L) increased both [Ca 2+ ] v and noradrenaline release but reduced force development. Acetylcholine (10 µmol/L) caused atropine-sensitive inhibition of [Ca 2+ ] v , noradrenaline release and force. In the presence of ω-conotoxin-GVIA, acetylcholine caused a further inhibition of all parameters., Conclusion: Modification of [Ca 2+ ] in arterial sympathetic axons and Schwann cells was assessed separately. K V 3.1 channels may be important regulators of [Ca 2+ ] v , noradrenaline release and force development. Presynaptic adrenoceptor and muscarinic receptor activation modify transmitter release through modification of [Ca 2+ ] v ., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Perivascular adipose tissue (PVAT) has been shown to produce vasoactive substances and regulate vascular tone. This function of PVAT has been reported to be altered in hypertension. However, the underlying mechanisms are not fully understood. In this study we used age-matched normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) as well as Sprague-Dawley rats and tested effects of PVAT on mesenteric small arteries. Vessels were mounted in a Mulvany-Halpern myograph and cumulative concentration-response relations to noradrenaline were determined in the presence or absence of PVAT. We found that PVAT has an anti-contractile effect on mesenteric small vessels, irrespective of strains. A reduced effect of PVAT was observed in SHR compared to WKY rats; the difference between strains was eliminated by 10 μM XE991, a blocker of Kv7 (KCNQ) voltage-dependent potassium channels. The anti-contractile effect of PVAT was not affected by depolarizing smooth muscle cells with high K(+) solution. Sensitivities to exogenous vasodilators acetylcholine or sodium nitroprusside were not potentiated but reduced in vessels with PVAT. Our results suggest that the reduced anti-contractile effect of PVAT in SHR correlates with a deficiency in Kv7 channels. Diffusion hindrance of PVAT is also a factor that should be considered in investigations on rat mesenteric small arteries., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of the present study was to characterize the function of resistance arteries, and the aorta, in rats with adenine-induced chronic renal failure (A-CRF). Sprague-Dawley rats were randomized to chow with or without adenine supplementation. After 6-10 wk, mesenteric arteries and thoracic aortas were analyzed ex vivo by wire myography. Plasma creatinine concentrations were elevated twofold at 2 wk, and eight-fold at the time of death in A-CRF animals. Ambulatory systolic and diastolic blood pressures measured by radiotelemetry were significantly elevated in A-CRF animals from week 3 and onward. At death, A-CRF animals had anemia, hyperphosphatemia, hyperparathyroidism, and elevated plasma levels of asymmetric dimethylarginine and oxidative stress markers. There were no significant differences between groups in the sensitivity, or maximal response, to ACh, sodium nitroprusside (SNP), norepinephrine, or phenylephrine in either mesenteric arteries or aortas. However, in A-CRF animals, the rate of aortic relaxation was significantly reduced following washout of KCl (both in intact and endothelium-denuded aorta) and in response to ACh and SNP. Also the rate of contraction in response to KCl was significantly reduced in A-CRF animals both in mesenteric arteries and aortas. The media of A-CRF aortas was thickened and showed focal areas of fragmented elastic lamellae and disorganized smooth muscle cells. No vascular calcifications could be detected. These results indicate that severe renal failure for a duration of less than 10 wk in this model primarily affects the aorta and mainly slows the rate of relaxation.

The possibility that Ca(2+)-activated Cl(-) conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I (Cl(Ca))) and cGMP-dependent (I (Cl(Ca,cGMP))) chloride conductances are important. The effect of anion substitution and Cl(-) channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl(-) (o) replacement with aspartate blocked vasomotion while 36mM SCN(-) (o) (substituted for Cl(-)) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca(2+)](i) disappeared with 36mM SCN(-). DIDS and Zn(2+) blocked I (Cl(Ca,cGMP)) but not I (Cl(Ca)). Vasomotion was not sensitive to DIDS and Zn(2+), and DIDS and Zn(2+) induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn(2+) was sensitive to 36mM SCN(-) (o). The anion substitution data indicate that Cl(-) is crucial for the V (m) and [Ca(2+)](i) oscillations underlying vasomotion. The Cl(-) channel blocker data are consistent with both CaCCs being important.

Unlabelled: Type 2 diabetes is associated with many circulatory manifestations, including alteration in endothelial function and hypertension. In this study we investigate the morphology and contractile response as well as the endothelial function of resistance arteries from the spontaneously diabetic Goto-Kakizaki (GK) rat, a model of lean type 2 diabetes expressing glucose intolerance., Methods: Isolated mesenteric small arteries were investigated under isometric conditions in a wire myograph system using noradrenaline (NA) and the endothelium-dependent vasorelaxant acetylcholine (ACh). Media thickness was measured and media lumen ratio calculated., Results: No apparent morphological difference was noted between the arteries from GK rats and control Wistar (CW) rats. When exposed to the maximal NA concentration used (30 microM), arteries from GK rats developed significantly more tension than arteries from CW rats. In the presence of indomethacin (a specific blocker of the COX synthase) and of L-NAME (an inhibitor of eNOS), the response to NA was still significantly greater in GK rat arteries. Under control conditions, arteries from both groups showed intact relaxation to ACh. After incubation with indomethacin and L-NAME, both groups showed a non-NO nonprostaglandin-dependent relaxation to ACh. This relaxation could be blocked by a combination of apamin and charybdotoxin., Conclusion: This study shows that mesenteric small arteries from the diabetic GK rat have increased contractile response to NA, along with a normal endothelial function and unaltered morphology.

The mechanisms leading to vasomotion in the presence of noradrenaline and inhibitors of the sarcoplasmic/endoplasmic reticulum calcium ATPase were investigated in isolated rat mesenteric small arteries. Isobaric diameter and isometric force were measured together with membrane potential in endothelial cells and smooth muscle cells (SMC). Calcium in the endothelial cells and SMC was imaged with confocal microscopy. In the presence of noradrenaline and cyclopiazonic acid, ryanodine-insensitive oscillations in tone were produced. The frequency was about 1 min(-1) and amplitude about 70% of the maximal tone. The amplitude was reduced by indomethacin and increased with L-NAME. Vasomotion was inhibited by nifedipine and by 40 mM potassium. The frequency was increased and amplitude decreased by removal of the endothelium and by application of charybdotoxin and apamin. The vasomotion was associated with in-phase oscillations of membrane potential in endothelial cells and SMC and oscillations of [Ca2+]i that were in near anti-phase. We suggest a working model for the generation of oscillation based on a membrane oscillator where ion channels in both endothelial cells and SMC interact via a current running between the two cell types through myoendothelial gap junctions, which sets up a near anti-phase oscillation of [Ca2+]i in the two cell types.

Synthetic peptides homologous to the extracellular loops of the major vascular connexins represent a novel class of gap junction blockers that have been used to assess the role of direct cellular communication in arteries and veins. However, the specificity of action of such peptides on the coupling between smooth muscle cells (SMCs) has not yet been fully characterized. Isolated third-order rat mesenteric arteries were therefore studied with respect to isometric tension (myography), intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ -sensitive dyes), membrane potential, and input resistance (sharp intracellular glass electrodes). Confocal imaging was used for visualization of [Ca2+]i events in individual SMCs in the arterial wall and membrane currents (patch clamp) measured in individual SMCs isolated from the same arteries. A triple peptide combination (37,43Gap 27 + 40Gap 27 + 43Gap 26) increased intercellular resistance (measured as input resistance) in intact arterial segments without affecting the membrane conductance of individual cells and also interrupted electrical coupling between pairs of rat aortic A7r5 myocytes. In intact arterial segments, the peptides desynchronized [Ca2+]i transients in individual SMCs and abolished vasomotion without suppressing Ca2+ transients in individual cells. They also depolarized SMCs, increased [Ca2+]i, and attenuated acetylcholine-induced, endothelium-dependent smooth muscle hyperpolarization. Experiments with endothelium-denuded arteries suggested that the depolarization produced by the peptides under basal conditions was in part secondary to electrical uncoupling of the endothelium from SMCs with loss of a tonic hyperpolarizing effect of the endothelium. Taken together, the results indicate that connexin-mimetic peptides block electrical signaling in rat mesenteric small arteries without exerting major nonjunctional effects.

Objective: We tested the hypothesis that cGMP can induce a state of only partial coordination of vascular smooth muscle cells (VSMC)., Methods: This was done by studying the concentration-dependent effect of 8Br-cGMP on isometric and isobaric force development of noradrenaline-activated segments of rat mesenteric small arteries in which the endothelium was removed. We further measured the concentration-dependent effect of 8Br-cGMP on VSMC membrane potential, spatially resolved [Ca(2+)](i) and VSMC membrane conductance., Results: With 300 microM 8Br-cGMP, coordinated [Ca(2+)](i) activity and vasomotion were seen as previously reported. At 10-30 microM 8Br-cGMP, beating isometric tension oscillations were seen. Isobaric recordings revealed oscillations with different frequencies in different parts of the arteries. At these (10-30 microM) 8Br-cGMP concentrations, membrane potential oscillations did not always concur with isometric tension oscillations, and [Ca(2+)](i) oscillations were only synchronized locally within groups of cells. 8Br-cGMP concentration-dependently decreased the frequency of vasomotion and, in unsynchronized hyperpolarized VSMC, the frequency of [Ca(2+)](i) waves., Conclusion: Our results demonstrated that cGMP can cause a partial coordination of the VSMC in the vascular wall (and at high concentrations near complete coordination). Furthermore, the cGMP concentration-dependent decrease of Ca(2+) wave frequency and of vasomotion frequency suggests that cGMP modifies oscillatory Ca(2+) release from the sarcoplasmic reticulum and supports the suggestion that this oscillatory release paces vasomotion., (Copyright 2005 S. Karger AG, Basel.)

Several different rat models have been developed for both type 1 and type 2 diabetes with the aim of displaying specific traits of diabetes. For example, a review on nephropathy associated with type 2 diabetes included 16 different rodent models ; new models are still being developed. The large number of different models developed for different traits makes it difficult to choose the right model for a given study. It is often a problem that the models are not sufficiently characterized, which makes it easy to misinterpret data or even come to the wrong conclusions. In this brief review, we will concentrate on the functional responses obtained in vitro from mesenteric arteries and aortic segments from rat models of diabetes. Since it is beyond the scope of this review to overview all different rodent models of diabetes, we will focus on two commonly used models of diabetes, namely the streptozotocin (STZ)-induced type 1 diabetic rat model and the inbred type 2 diabetic Goto-Kakizaki (GK)-rat model.

Arteries display cyclic diameter variations, vasomotion. In vivo, these rhythmic contractions are modulated by the influence of sympathetic nerves. In this study, we investigated the effect of burst stimulation of intramural nerves in vitro on the vasomotion of rat mesenteric small arteries. Vessels were mounted for isometric force measurement. After initiation of vasomotion with noradrenaline (0.5-2 microM), periarterial sympathetic nerves were stimulated electrically (10 impulses at 20 Hz) at approximately half-minute intervals. With a delay of 2-3 s, a neurogenic burst caused a brief contraction of the vascular smooth muscle and altered the period of the current vasomotion cycle. The effect on amplitude decayed rapidly and was practically not apparent in the next vasomotion cycle after the burst. With respect to period, stimulation at increasing intervals from the trough in force of vasomotion caused gradual prolongation of the cycle until a critical interval was reached, after which cycle duration was reduced instead. Since subsequent cycles were not affected, a change in phase remained. When two segments of oscillating arteries were mounted in a two-vessel myograph, simultaneously applied bursts of impulses synchronized their oscillation. The data suggest that changes in neural activity are able to make different vessels oscillate in phase, thereby coordinating vasomotion in different parts of the vascular tree, possibly explaining the synchronicity of vasomotion in different vascular beds that can be observed in vivo., (Copyright 2005 S. Karger AG, Basel.)

1 Heptanol, 18alpha-glycyrrhetinic acid (18alphaGA) and 18beta-glycyrrhetinic acid (18betaGA) are known blockers of gap junctions, and are often used in vascular studies. However, actions unrelated to gap junction block have been repeatedly suggested in the literature for these compounds. We report here the findings from a comprehensive study of these compounds in the arterial wall. 2 Rat isolated mesenteric small arteries were studied with respect to isometric tension (myography), [Ca2+]i (Ca(2+)-sensitive dyes), membrane potential and--as a measure of intercellular coupling--input resistance (sharp intracellular glass electrodes). Also, membrane currents (patch-clamp) were measured in isolated smooth muscle cells (SMCs). Confocal imaging was used for visualisation of [Ca2+]i events in single SMCs in the arterial wall. 3 Heptanol (150 microm) activated potassium currents, hyperpolarised the membrane, inhibited the Ca2+ current, and reduced [Ca2+]i and tension, but had little effect on input resistance. Only at concentrations above 200 microm did heptanol elevate input resistance, desynchronise SMCs and abolish vasomotion. 4 18betaGA (30 microm) not only increased input resistance and desynchronised SMCs but also had nonjunctional effects on membrane currents. 18alphaGA (100 microm) had no significant effects on tension, [Ca2+]i, total membrane current and synchronisation in vascular smooth muscle. 5 We conclude that in mesenteric small arteries, heptanol and 18betaGA have important nonjunctional effects at concentrations where they have little or no effect on intercellular communication. Thus, the effects of heptanol and 18betaGA on vascular function cannot be interpreted as being caused only by effects on gap junctions. 18alphaGA apparently does not block communication between SMCs in these arteries, although an effect on myoendothelial gap junctions cannot be excluded.

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 microM BAY K8644, 0.4 microM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 microM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN- > Br- > I- > Cl- > acetate > F- >> aspartate, but the conductance sequence was I- > Br- > Cl- > acetate > F- > aspartate = SCN-. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.

A possible role for a metabolite of cytochrome P-450 omega-hydroxylase in the initial and sustained phases of the myogenic response in cannulated rat mesenteric small arteries was studied. With slight preconstriction (norepinephrine and neuropeptide Y), pressure was raised from 60 to 100 mmHg, and both initial (within 2 min) and sustained phases (at 10 min) of the myogenic response were quantified. The myogenic response was fully inhibited by D600 (methoxyverapamil). Ketoconazole and 17-octadecanoic acid did not affect the initial phase but inhibited the sustained phase. In contrast, miconazole did not affect either phase. Charybdotoxin and iberiotoxin potentiated the initial phase but eliminated the sustained phase. Apamin, glibenclamide, 4-aminopyridine, and barium had no effect on either phase. The results demonstrate different mechanisms for the initial and sustained phases of the myogenic response of rat mesenteric small arteries. Only the sustained phase appears mediated through a cytochrome P-450 omega-hydroxylase metabolite and calcium-activated K+ channels. However, both phases of the response are dependent on calcium influx through voltage-dependent calcium channels.

Simultaneous measurements of intracellular calcium concentration ([Ca(2+)](i)) and tension were performed to clarify whether the mechanisms which cause the neuropeptide Y (NPY)-elicited contraction and potentiation of noradrenaline contractions, and the NPY inhibition of forskolin responses are linked to a single or different NPY receptor(s) in rat mesenteric small arteries. In resting arteries, NPY moderately elevated [Ca(2+)](i) and tension. These effects were antagonized by the selective Y(1) receptor antagonist, (R)-N(2)-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]-D-argininea mide (BIBP 3226) (apparent pK(B) values of 8.54+/-0.25 and 8.27+/-0.17, respectively). NPY (0.1 microM) caused a near 3 fold increase in sensitivity to noradrenaline but did not significantly modify the tension-[Ca(2+)](i) relationship for this agonist. BIBP 3226 competitively antagonized the contractile response to NPY in arteries submaximally preconstricted with noradrenaline (pA(2) 7.87+/-0.20). In arteries activated by vasopressin, the adenylyl cyclase activator forskolin (3 microM) induced a maximum relaxation and a return of [Ca(2+)](i) to resting levels. NPY completely inhibited these effects. The contractile responses to NPY in arteries maximally relaxed with either sodium nitroprusside (SNP) or nifedipine were not significantly higher than those evoked by the peptide at resting tension, in contrast to the contractions to NPY in forskolin-relaxed arteries. BIBP 3226 competitively antagonized the contraction to NPY in forskolin-relaxed arteries with a pA(2) of 7.92+/-0.29. Electrical field stimulation (EFS) at 8-32 Hz caused large contractions in arteries relaxed with either forskolin or noradrenaline in the presence of phentolamine. These responses to EFS were inhibited by BIBP 3226. Similar EFS in resting, non-activated arteries did not produce any response. The present results suggest that different intracellular pathways are linked to a single NPY Y(1) receptor in intact rat mesenteric small arteries, and provide little support for involvement of other postjunctional NPY receptors in the contractile responses to NPY. Neurally released NPY also seems to act through Y(1) receptors, and may serve primarily as an inhibitor of vasodilatation.

1. Mechanisms of Ca2+ sensitization of force production by noradrenaline were investigated by measuring contractile responses, intracellular Ca2+ concentration ([Ca2+]i) and phosphorylation of the myosin light chain (MLC) in intact and alpha-toxin-permeabilized rat mesenteric small arteries. 2. The effects of noradrenaline were investigated at constant membrane potential by comparing fully depolarized intact arteries in the absence and presence of noradrenaline. Contractile responses to K-PSS (125 mM K+) and NA-K-PSS (K-PSS + 10 microM noradrenaline) were titrated to 30 and 75%, respectively, of control force, by adjusting extracellular Ca2+ ([Ca2+]o). At both force levels, [Ca2+]i was substantially lower with NA-K-PSS than with K-PSS. With K-PSS, the proportion of MLC phosphorylated (approximately 30%) was similar at 30 and 75% of control force; with NA-K-PSS, MLC phosphorylation was greater at the higher force level (40 vs. 34%). 3. In alpha-toxin-permeabilized arteries, the force response to 1 microM Ca2+ was increased by 10 microM noradrenaline, and MLC phosphorylation was increased from 35 to 45%. The protein kinase C (PKC) inhibitor calphostin C (100 nM) abolished the noradrenaline-induced increase in MLC phosphorylation and contractile response, without affecting the contraction in response to Ca2+. Treatment with ATP gamma S in the presence of the MLC kinase inhibitor ML-9 increased the sensitivity to Ca2+ and abolished the response to noradrenaline. 4. The present results show that that in rat mesenteric small arteries noradrenaline-induced Ca2+ sensitization is associated with an increased proportion of phosphorylated MLC. The results are consistent with a decreased MLC phosphatase activity mediated through PKC. Furthermore, while MLC phosphorylation is a requirement for force production, the results show that other factors are also involved in force regulation.

We have investigated the effect of intracellular calcium levels for membrane potential during noradrenaline application in isolated small arteries. Rat mesenteric small arteries were mounted for isometric tension measurement. Smooth muscle membrane potentials were measured by conventional intracellular electrodes, and intracellular calcium concentration was measured using Fura-2 fluorescence. Under control conditions, noradrenaline caused contraction and depolarization from -55.5 to -29.3 mV. In intact arteries, depleting intracellular calcium stores with thapsigargin caused smooth muscle hyperpolarization and inhibited contraction to noradrenaline. In de-endothelialized vessels, thapsigargin still depleted calcium stores, but did not affect either the depolarization or contraction caused by noradrenaline. In noradrenaline-activated vessels, inhibition of calcium influx by amlodipine caused tension and calcium levels to fall to near-baseline levels, but membrane potential returned by only 55%. Treatment with a combination of thapsigargin, D-600 and BAPTA-AM inhibited the tension and calcium responses to noradrenaline, but the membrane potential response was reduced by only 34%. Acute reduction of extracellular chloride concentration caused similar, small depolarization at rest and during noradrenaline exposure. It is concluded that an elevation of intracellular calcium concentration is not essential for noradrenaline depolarization, although part of the depolarization is associated with the raised intracellular calcium level.

1. Simultaneous measurements of membrane potential and tension were performed to investigate the intracellular mechanisms of neuropeptide Y (NPY) in rat mesenteric small arteries. 2. NPY (0.1 microM) depolarized arterial smooth muscle cells from -55 to -47 mV and increased wall tension by 0.22 N m-1, representing 11% of the contraction elicited by a high-potassium solution. Isoprenaline (1 microM) and acetylcholine (1 microM) evoked hyperpolarizations of 11 and 17 mV, respectively. NPY inhibited the isoprenaline-induced effects on membrane potential without affecting those of acetylcholine. 3. Forskolin evoked sustained concentration-dependent hyperpolarizations of small mesenteric arteries. NPY (0.1 microM) inhibited the responses to 1 microM forskolin, but did not alter the stable hyperpolarization elicited by the specific activator of protein kinase A (PKA) SP-5,6-DCl-cBIMPS (0.1 mM). Forskolin increased the cyclic AMP (cAMP) content of the arteries 21-fold, and NPY inhibited the forskolin-evoked increase in cAMP levels by 91%. 4. The hyperpolarization produced by 1 microM forskolin was not affected by either charybdotoxin (0.1 microM) or 4-aminopyridine (0.5 mM), but glibenclamide (5 microM) inhibited the hyperpolarization by 70%. Glibenclamide also inhibited the hyperpolarization evoked by SP-5,6-DCl-cBIMPS by 59%. 5. Neither depolarization nor contraction caused by NPY were significantly affected by either glibenclamide (5 microM) or nifedipine (1 microM), but they were reduced by gadolinium (10 microM). However, the blocking effect of NPY on forskolin-elicited hyperpolarization was not affected by gadolinium. 6. Charybdotoxin (0.1 microM) and 4-aminopyridine (0.5 mM) strongly enhanced the depolarization and contraction caused by NPY (0.1 microM), and nifedipine (1 microM) prevented the enhanced responses to NPY in the presence of charybdotoxin. 7. These findings suggest that NPY acts through at least two different intracellular mechanisms in mesenteric small arteries: a depolarization of arterial smooth muscle which is probably due to activation of non-selective cation channels, and a marked inhibition of adenylate cyclase activity, which in turn inhibits the hyperpolarization produced by cAMP accumulation in these arteries.

In small blood vessels, elevation of transmural pressure induces myogenic constrictions and smooth muscle depolarization. The role of calcium-activated K+ channels (KCa channels) in these responses was examined in cannulated rat mesenteric small arteries. Inner and outer diameter were continuously monitored with a video technique. Smooth muscle membrane potential was recorded simultaneously using microelectrodes. To test for myogenic responsiveness, the transmural pressure was changed stepwise in the range between 10 and 120 mmHg. Pressure elevation induced moderate myogenic responses and significant depolarization, from -54.5 +/- 0.4 (SE) mV (n = 56) at 10 mmHg to -47.3 +/- 1.8 mV (n = 12) at 120 mmHg. Norepinephrine (NE, 0.67 and 10 microM) induced constriction and vasomotion, augmented myogenic responsiveness, and shifted the pressure-membrane potential relation to more depolarized values. Blockade of the Kca channels with charybdotoxin (ChTX) suppressed the responsiveness to pressure. In the absence of ChTX, with 0.67 microM NE, pressure elevation from 10 to 120 mmHg induced depolarization from -46.9 +/- 1.0 (n = 16) to -35.8 +/- 0.7 (n = 12) mV, whereas because of the myogenic response, the diameter increased only by 7%. In the presence of ChTX, with 0.3 microM NE, pressure changed the membrane potential from -41.0 +/- 1.1 (n = 12) to -37.8 +/- 0.7 mV (n = 4), which was not significant, and the diameter increased by 28%. These results demonstrate that blockade of KCa channels reduces responsiveness to pressure elevation. This suggests that pressure may induce depolarization and concomitant myogenic responsiveness by closure of KCa channels.

The role of membrane potential-dependent and independent regulation of the intracellular free calcium concentration ([Ca2+]i) was assessed in the mesenteric small arteries of Wistar rats. [Ca2+]i was determined by Fura-2 fluorescence. Membrane potential measurements were made using intracellular microelectrodes. Depolarization with a high-potassium solution (K-PSS) elevated [Ca2+]i and induced contraction. Further addition of 10 microM noradrenaline (NA) did not elevate [Ca2+]i further but enhanced tone. Addition of calcium channel inhibitors (felodipine or D-600) inhibited the maintained rise in [Ca2+]i with K-PSS, but NA still elevated [Ca2+]i and force to about half the previous level. Further addition of either ryanodine or thapsigargin eliminated the rise in [Ca2+]i with NA, although 10-20% of the contraction remained. Simultaneous measurements of membrane potential, [Ca2+]i, and force during cumulative additions of NA or K-PSS in the absence of inhibitors showed similar relations between membrane potential and [Ca2+]i for each means of activation. The results indicate that membrane potential and [Ca2+]i are strongly correlated in mesenteric small arteries. A small part of the [Ca2+]i increase to NA can be attributed to release from intracellular stores. Membrane potential-independent calcium channels that are directly operated by adrenoceptors appear to play a minor role in the regulation of [Ca2+]i in these vessels.

The present study was undertaken to examine the influence of transmural pressure on vasomotion and to determine if any such influence was endothelium-dependent. Responses to changes in intravascular pressure of cannulated mesenteric small arteries were investigated under no-flow conditions. Both intact and endothelium-denuded arteries dilated passively when intravascular pressure was increased stepwise from 20 to 140 mmHg. When tone was induced by noradrenaline, pressure increase resulted only in dilatation, independent of endothelium. The sensitivity to noradrenaline was significantly increased in vessels without endothelium, indicating a relaxing influence of the endothelium. Rhythmic contractions in response to noradrenaline occurred in all intact arteries, but were absent when the endothelium was removed. The amplitude of the rhythmic contractions decreased significantly when transmural pressure was elevated. The frequency increased when pressure was elevated from 20 to 80 mmHg and then remained rather constant during further pressure increases. As shown previously in non-pressurized arteries, exogenous cyclic GMP induced oscillations in endothelium-denuded arteries. Pressure-related effects on vasomotion were not dependent on an intact endothelium. Ryanodine, ouabain or verapamil inhibited the rhythmic activity, confirming previous results in non-pressurized arteries. Thus, changes in transmural pressure can modulate vasomotion, but this effect does not appear to be mediated by the endothelium. Generation of vasomotion may depend on release of Ca2+ from intracellular stores, the activity of the Na+, K(+)-pump and transmembrane Ca2+ inflow in a pressurized artery as shown previously in these arteries under isometric conditions.

Free cytosolic Ca2+ was measured with sub-micrometer-tip, double-barrelled, Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries. The purpose was to establish intracellular free Ca2+ concentration ([Ca2+]i) values in resting and stimulated vessels. Isolated vessels were mounted for isometric force measurements. Measured with electrodes, mean [Ca2+]i was 115 and 708 nM under resting and norepinephrine-activated conditions, respectively. Fura 2 was calibrated intracellularly including determination of the intracellular dissociation constant (Kd) of the fura 2:Ca2+ complex. The intracellular Kd was 342 nM. With this value of Kd, fura 2 measurements of mean [Ca2+]i were 129 and 537 nM under resting and norepinephrine-activated conditions, respectively. The values measured with the two techniques were thus in good accordance.

The concentration dependence of ATP-induced contractions in isolated resistance arteries was estimated using photolysis of caged ATP. Rat mesenteric vessels were isolated and mounted for force registration in a small chamber allowing illumination from a xenon-flash lamp. Photolysis of 100 microM caged ATP, which released about 20 microM ATP within a few milliseconds in the vessel, induced a transient contraction with an amplitude approximately 40-50% of the response induced by 10 microM noradrenaline. The responses could neither be induced by the light flash as such nor by caged ATP alone nor by photolysis of caged phosphate. The amplitude of the contractions was dependent on the concentration of caged ATP, and the effective concentration for ATP was estimated to be in the range of 1-10 microM. In contrast, when ATP was introduced by diffusion, about a 100-fold higher concentration was required. Thus photolytic release of ATP minimizes metabolism before its action on receptors and reveals action of ATP in a concentration range consistent with a role of ATP as a transmitter in nervous regulation of the tone of resistance vessels.

We have previously studied the transmitters involved in, and the calcium dependency of, the neurogenic response to continuous, regular nerve stimulation in small mesenteric arteries from the rat. We have now studied responses to irregular nerve stimulation in these respects. Small mesenteric arteries from rat were mounted into a myograph and the intramural sympathetic nerves were activated by field stimulation. Irregular nerve stimulation was patterned after recorded activity in human cutaneous nerves. The response to irregular stimulation was reduced by only approximately 50% in the presence of the alpha 1-adrenoceptor antagonist prazosin. In particular, responses to low-frequency stimulation were resistant to prazosin. When the extracellular calcium concentration was reduced from 2.5 to 1.0 mM, approximately 50% of the response to irregular nerve stimulation remained. Responses to low-frequency stimulation were particularly reduced. Thus in these arteries the neurogenic response is caused by noradrenaline and a, perhaps purinergic, co-transmitter. The co-transmitter is important for the response to low-frequency stimulation and for the initial part of the contraction caused by high-frequency stimulation. Reducing the calcium concentration affects more strongly the response to low-frequency nerve stimulation.

Contractile responses to electrical field stimulation of excised small mesenteric arteries and veins of the rat were compared when stimuli were delivered in irregular bursts or at regular intervals. Spontaneously occurring skin vasoconstrictor impulses in a few-unit median nerve recording in man were stored on tape and used to trigger a stimulator. Two irregular stimulation sequences at average frequencies of 1.6 and 1.8 Hz, respectively, were used. In the arteries, average contractile responses were significantly greater at an irregular than at an even stimulation frequency, but in the veins, similar degrees of contraction were obtained with the two modes of stimulation. The frequency-response relationships to continuous regular stimulation showed the artery to respond less than the vein at low frequencies. This apparently explains the differences in behaviour between the vessels to irregular stimulation. The results show that not only the number of impulses, but also their pattern of occurrence, may influence the degree of vasoconstriction. Thus, the normal irregular sympathetic discharge pattern in itself has a bearing on the physiology of neuro-effector control mechanisms.

The role of the prejunctional alpha 2-receptors for the response to vaso-constrictor nerve stimulation has been examined before and after inhibition of neuronal transmitter reuptake in mesenteric resistance vessels from the rat. Small arteries (diameter about 200 micron) feeding the jejunum were mounted in a myograph for recording of their isometric wall tension during transmural field stimulation of the intramural nerves. Blockade of prejunctional alpha 2-receptors with 0.01 microM idazoxan (RX 781094) caused a marginal potentiation of the neurogenic response when neuronal reuptake was left intact. Also, inhibition of reuptake alone with 3 microM cocaine had little effect on the response. However, when both alpha 2-receptors and reuptake had been inhibited, a strong enhancement of the neurogenic vasoconstriction was observed. Similar findings were made when yohimbine and LU 3-010 instead were used for alpha 2-blockade and reuptake inhibition, respectively. The results thus indicate that in these resistance arteries the effector response is normally influenced by the combined activity of alpha 2-receptors and uptake, and that failure of one mechanism increases the activity of the other, so as to maintain a largely constant effector response.

In the present study we examine the calcium requirements of the neurogenic response in vitro of small arteries (150-200 microns diameter) from the mesentery of Wistar rats. Intramural nerves were activated with electrical field stimulation. Responses to single impulses and to low-frequency repeated stimulation were reduced or abolished by reducing the Ca2+ concentration in the bathing solution from 2.5 to 1.0 mM. Responses to higher frequencies (16 Hz) were only slightly affected. Since calcium reduction had markedly less effect on responses to direct activation of the smooth muscle and on responses to any dose of exogenous noradrenaline, the calcium reduction had mainly pre-junctional effects. The data show that part of the neurogenic response is highly calcium-sensitive, perhaps more so than would be expected of a purely noradrenergic transmission.

The effects of 30 minutes of exposure to ouabain on calcium sensitivity have been investigated in two types of resistance vessels from 12 pairs of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Branches of the superior mesenteric and femoral arteries, with internal diameters of about 200 micrometer, were mounted as ring preparations in a myograph capable of measuring their isometric wall tension. Dose-response curves for calcium upon norepinephrine stimulation were determined under conditions where neuronal uptake was eliminated. Initially, when stimulated with norepinephrine, the SHR vessels from both locations were more sensitive to calcium and had stronger contractions than their controls. The addition of ouabain (1 mM) to the relaxed vessels immediately elicited a moderate, transient contraction in the branches of the femoral artery, whereas no response was observed in the mesenteric vessels. Although the addition of ouabain to activated vessels produced an immediate potentiation of the response, prolonged (30-minute) exposure to ouabain reduced active tension development upon norepinephrine stimulation in all vessels. The reduction was greatest in the SHR vessels, so that, under these conditions, the norepinephrine-activated calcium sensitivity of corresponding SHR and WKY vessels was similar. By contrast, responses to norepinephrine in high potassium solution were unaffected. The results suggest that under normal conditions, SHR vessels may have a specific increase in the permeability of the norepinephrine-activated calcium channels. Prolonged exposure to ouabain appears to reduce the permeability of these channels, providing an explanation for why this treatment eliminates the difference in calcium sensitivity of the SHR and WKY vessels.

The authors have previously shown that vasoconstrictor nerve responses of mesenteric small arteries from spontaneously hypertensive rats (SHR) are increased compared with normotensive rats. In this paper, whether this is due to differences in the degree of negative feedback of the transmitter on its own release is investigated. Small (i.d. 200 microns) arteries from the mesenteric arcades of SHR and normotensive control rats (NCR) were mounted in a Mulvany-Halpern myograph. In both SHR and NCR, nerve stimulation and applied noradrenaline elicited maximal contractions of similar magnitude, although SHR vessels were somewhat stronger and more responsive to nerve stimulation. Blocking alpha 2-receptors with 10 nM RX 781094 reduced the frequency for half-maximal activation to 84% and 80%, respectively, but did not eliminate the difference between the strains. Presynaptic alpha 2-receptors thus do not seem to cause the enhanced vasoconstrictor responses in SHR vessels.

Experiments on the vasoconstrictor fiber control of isolated spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) resistance vessels, which is throughout more efficient in SHR, suggest that the presynaptic alpha 2-negative feedback mechanism and the reuptake pump are largely complementary in adjusting the effective transmitter concentrations in these narrow junctions, and first when both are blocked a marked leftward displacement of the nervous frequency-response curve ensues. Also, ouabain causes a marked leftward displacement of the frequency response curve in a way which suggests a considerable increase of transmitter release/impulse. Finally, normal rat plasma contains agent(s) which, even in very low concentrations, seems to cause a strong ouabain-like effect on the neurogenic responses. However, these various interferences with the neurogenic vascular effects influence SHR and WKY to largely equal extents, implying that the more efficient nervous control in SHR vessels is not merely due to an altered balance of the ordinary local mechanisms that influence the adrenergic transmitter release.

The effects of distension on the sensitivity to noradrenaline and potassium were assessed in ring preparations of 200 micron branches from the superior mesenteric artery of the rat. In response to noradrenaline, maximal tension development occurred at smaller luminal diameters than with potassium activation. The sensitivity to noradrenaline increased with increasing distension whereas potassium sensitivity remained largely unchanged. A distension-dependent increase in noradrenaline sensitivity was observed also in vessels depolarized with high-potassium solution. The role of the distension-dependent sensitivity to noradrenaline for isotonic contractions was also studied. Upon activation with applied noradrenaline, submaximal isotonic responses were somewhat smaller than isometric responses at the same dose. This may be ascribed to a reduction in sensitivity during the isotonic contraction. Upon neurogenic activation, no difference between isotonic and isometric responses was found. The results show that distension increases noradrenaline sensitivity in resistance vessels in vitro, but that the consequences for their function as neuroeffectors may be small.

We have previously shown that the contraction of small mesenteric arteries in response to nerve stimulation is enhanced by preceding high-frequency stimulation. We have now investigated the calcium dependency of this post-stimulatory potentiation. Small arteries (inner diameter 150-250 microns) from normotensive rats were dissected free from surrounding tissue, and segments were mounted in a myograph where the wall tension was measured at well-defined circumferences. Nerve stimulation was performed by field stimulation. A single stimulation of the nerve caused a contraction of 2.6 +/- 0.25% of maximal adrenergic response. After a high-frequency nerve stimulation with 16 Hz and 480 pulses the response to a single nerve stimulation was enhanced 6.6 +/- 1.3 times. The potentiation decayed with a time constant of 93.7 +/- 20.0 s. The amplitude of the post-stimulatory potentiation was dependent on the extracellular calcium concentration during the conditioning stimulation. In a solution containing 2.5 mM calcium the single twitch was enhanced 6.6 times while after exposure to reduced calcium (0.5 mM) it was only enhanced twice. The contraction caused by a short burst of high-frequency nerve stimulation (20 Hz and 10 pulses) was potentiated four times by a conditioning stimulation (16 Hz and 480 pulses), and this potentiation seemed to be independent of the extracellular calcium concentration during the conditioning stimulation. Thus the magnitude of the post-stimulatory potentiation of single nerve stimulations is linearly related to the extracellular calcium concentration during the conditioning nerve stimulation. For the potentiation of the response to burst stimulation no relation was found between the potentiation and extracellular calcium.